When DNA is replicated, the chromatin structure must be duplicated, as well. The pre-existing (or parental) histones are recycled and then reassembled following the passage of the replication near their original site of residence. Hence, the epigenetic inheritance of a specific chromatin domain can be facilitated by the spatial retention of the correctly modified parental histones. However, packaging of the newly replicated DNA requires the incorporation of an equal quantity of newly synthesized histones. This complicates the situation as, rather than being a blank slate upon which these specific patterns of modification can be reproduced, the newly synthesized histones are assembled with precise patterns of modification. The goal of this proposal is to understand the function of the histone acetyltransferase Hat1, which is necessary for the acetylation of newly synthesized histones. This proposal will focus on the function of Hat1 in the assembly and regulation of chromatin structure, as well as other aspects of cell metabolism. The first specific aim will use a variety of in vitro analyses to examine the mechanisms by which Hat1 influences the modification of newly synthesized histones and replication-coupled chromatin assembly. Hat1- containing complexes have both histone acetyltransferase and histone chaperone activities. One sub-aim will generate Hat1 mutations that specifically block each of these activities to identify the functions of Hat1 that are important for the processing of newly synthesized H3 and H4. A second sub-aim will employ affinity purification of newly replicated DNA to identify Hat1-dependent changes in nascent chromatin. The goal of the second aim of this proposal is to determine the role of Hat1 in mitochondrial function. Protein acetylation is known to play a critical role in the regulation of mitochondrial activity. However, the protein acetyltransferases responsible for the acetylation of mitochondrial proteins are completely unknown. We have found that Hat1 is localized to mitochondria and the goal of this aim is to identify substrates of hat1 in this organelle and determine how Hat1-dependent mitochondrial acetylation affects the function of this organelle.